Barth Syndrome (BTHS) is a rare X-linked genetic disorder with an incidence of 1/300,000 live births. Clinical manifestations include dilated cardiomyopathy, skeletal myopathy, neutropenia, 3-methylglutaconic aciduria, severe growth retardation, and moderate learning disabilities. In BTHS, the myopathy arises from a global mitochondrial defect. BTHS patients have mutations in the tafazzin gene (TAZ) which belongs to a family of established and putative glycerolipid acyltransferases. Consistent with this finding is that tissues from BTHS patients contain decreased cardiolipin (CL), a membrane lipid essential for proper mitochondrial function. To date, published evidence for a direct role of TAZ in CL biosynthesis is strong, but circumstantial. In fact, our studies show that recombinant TAZ fails to exhibit monolysocardiolipin acyltransferase activity, a critical step in CL remodeling. We propose to take an innovative approach and determine the TAZ-mediated changes in glycerolipids. We will use the information about altered lipid profiles as valuable clues to identify the enzymatic function of TAZ. Our data show TAZ-induced increases in fatty acid incorporation into phosphatidylglycerol (PG) and decreases into phosphatidic acid (PA). TAZ also interferes with PA formation in direct assays. Finally, we have shown that absence of TAZ decreases bis(monoacylglycerol)phosphate (BMP) and causes accumulation of acylphosphatidylglycerol (APG). We hypothesize that tafazzin a) may function early in the glycerolipid pathway at the step of PA formation or use, and b) may play a role in APG and BMP synthesis. We will use ESI/MS to determine changes in the entire lipid profile of cells overexpressing TAZ and in BTHS lymphoblasts. We will identify the enzymatic defect in BTHS by assaying recombinant TAZ for activities in the glycerolipid pathway. Our rationale for this proposal is that determining TAZ function furthers our understanding of the BTHS defect and provides critical knowledge about mechanisms by which abnormal lipid metabolism disrupt normal cellular functions.